Polymeric oligonucleotide prodrugs

ABSTRACT

Polymer conjugates containing nucleotides and/or oligonucleotides are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from U.S.Provisional Patent Application Serial No. 60/462,070, filed Apr. 13,2003, the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present inventions relates to polymeric oligonucleotideprodrugs useful as therapeutic agents. Compositions and methods of usingsuch prodrugs are also provided.

BACKGROUND

[0003] It is well known that most of the bodily states in multicellularorganisms, including most disease states, are effected by proteins. Suchproteins, either acting directly or through their enzymatic or otherfunctions, contribute in major proportion to many diseases andregulatory functions in animals and man. For disease states, classicaltherapeutics have generally focused upon interactions with such proteinsin efforts to moderate their disease-causing or disease potentiatingfunctions. In newer therapeutic approaches, modulation of the actualproduction of such proteins is desired. By interfering with theproduction of proteins, the maximum therapeutic effect may be obtainedwith minimal side effects. It is therefore a general object of suchtherapeutic approaches to interfere with or otherwise modulate geneexpression, which would lead to undesired protein formation.

[0004] One method for inhibiting specific gene expression is with theuse of oligonucleotides, especially oligonucleotides that arecomplementary to a specific target messenger RNA (mRNA) sequence.Generally, nucleic acid sequences complementary to the products of genetranscription (e.g., mRNA) are designated “antisense”, and nucleic acidsequences having the same sequence as the transcript or being producedas the transcript are designated “sense”. See, e.g., Crooke, 1992, Annu.Rev. Pharmacol. Toxicol., 32: 329-376. An antisense oligonucleotide canbe selected to hybridize to all or part of a gene, in such a way as tomodulate expression of the gene. Transcription factors interact withdouble-stranded DNA during regulation of transcription. Oligonucleotidescan serve as competitive inhibitors of transcription factors to modulatetheir action. Several recent reports describe such interactions (seeBielinska, A., et al., 1990, Science, 250: 997-1000; and Wu, H., et al.,1990, Gene 89: 203-209).

[0005] Molecular strategies are being developed to down-regulateunwanted gene expression. Recently, the use of modified oligonucleotidecompounds has developed into a promising method of treatment againstsuch diseases as viral infections, inflammatory and genetic disorder andsignificantly, cancer. Antisense DNAs were first conceived as alkylatingcomplementary oligodeoxynucleotides directed against naturally occurringnucleic acids (Belikova, et al., Tetrahedron Lett. 37:3557-3562, 1967).Zamecnik and Stephenson were the first to propose the use of syntheticantisense oligonucleotides for therapeutic purposes. (Zamecnik &Stephenson, 1978, Proc. Natl. Acad. Sci. U.S.A., 75:285-289; Zamecnik &Stephenson, 1978, Proc. Natl. Acad. Sci. U.S.A., 75:280-284). Theyreported that the use of an oligonucleotide 13-mer complementary to theRNA of Rous sarcoma virus inhibited the growth of the virus in cellculture. Since then, numerous other studies have been publishedmanifesting the in vitro efficacy of antisense oligonucleotideinhibition of viral growth, e.g., vesicular stomatitis viruses (Leonettiet al., 1988, Gene, 72:323), herpes simplex viruses (Smith et al., 1987,Proc. Natl. Acad. Sci. U.S.A. 83:2787), and influenza virus (Seroa; etal., 1987, Nucleic Acids Res. 15:9909).

[0006] Oligonucleotides have also found use in among others, diagnostictests, research reagents e.g. primers in PCR technology and otherlaboratory procedures. Oligonucleotides can be custom synthesized tocontain properties that are tailored to fit a desired use. Thus numerouschemical modifications have been introduced into oligomeric compounds toincrease their usefulness in diagnostics, as research reagents and astherapeutic entities.

[0007] Although oligonucleotides, especially antisense oligonucleotidesshow promise as therapeutic agents, they are very susceptible tonucleases and can be rapidly degraded before and after they enter thetarget cells making unmodified antisense oligonucleotides unsuitable foruse in in vivo systems. Because the enzymes responsible for thedegradation are found in most tissues, modifications to theoligonucleotides have been made in an attempt to stabilize the compoundsand remedy this problem. The most widely tested modifications have beenmade to the back-bone portion of the oligonucleotide compounds. Seegenerally Uhlmann and Peymann, 1990, Chemical Reviews 90, at pages545-561 and references cited therein. Among the many different backbones made, only phosphorothioate showed significant antisense activity.See for example, Padmapriya and Agrawal, 1993, Bioorg. & Med. Chem.Lett. 3, 761. While the introduction of sulfur atoms to the back boneslows the enzyme degradation rate, it also increases toxicity at thesame time. Another disadvantage of adding sulfur atoms is that itchanges the back bone from achiral to chiral and results in 2^(n)diastereomers. This may cause further side effects. Still moredisadvantages of present antisense oligonucleotides are that they maycarry a negative charge on the phosphate group which inhibits itsability to pass through the mainly lipophilic cell membrane. The longerthe compound remains outside the cell, the more degraded it becomesresulting in less active compound arriving at the target. A furtherdisadvantage of present antisense compounds is that oligonucleotidestend to form secondary and high-order solution structures. Once thesestructures are formed, they become targets of various enzymes, proteins,RNA, and DNA for binding. This results in nonspecific side effects andreduced amounts of active compound binding to mRNA. Other attempts toimprove oligonucleotide therapy have included adding a linking moietyand polyethylene glycol. See for example, Kawaguchi, et al., Stability,Specific Binding Activity, and Plasma Concentration in Mice of anOligodeoxynucleotide Modified at 5′-Terminal with Poly(ethylene glycol),Biol. Pharm. Bull., 18(3) 474-476 (1995), and U.S. Pat. No. 4,904,582.In both of these examples, the modifications involve the use of linkingmoieties that are permanent in nature in an effort to stabilize theoligonucleotide against degradation and increase cell permeability.However, both of these efforts fail to provide any efficacy.

[0008] Due to the inadequacies of the present methods, there exists aneed to improve stability and resistance to nuclease degradation as wellas decrease toxicity and increase binding affinity to mRNA ofoligonucleotide compounds. The current oligonucleotide therapy isexpensive. This is mainly due to the degradation problem. Thus, there isa real need to protect the antisense oligonucleotide compounds againstdegradation, prevent the formation of high-order structures and at thesame time deliver sufficient amounts of active antisense oligonucleotidecompounds to the target. This invention provides such improvements.

SUMMARY OF THE INVENTION

[0009] In one aspect of the invention there are provided oligonucleotideprodrugs of formula (I):

[0010] wherein:

[0011] R₁ and R₂ are independently H or a polymer residue;

[0012] L₁ and L₄ are independently selected releasable linking moieties;

[0013] L₂ and L₃ are independently selected spacing groups;

[0014] X₁ is a nucleotide residue or an oligonucleotide residue;

[0015] m, n, o and p are independently zero or a positive integer,provided that either (o+n) or (p+m)≧2.

[0016] Another aspect of the invention includes bifunctional compoundsthat are formed when R₁ and/or R₂ are polymer residues which includeboth an alpha and an omega terminal linking group as described herein sothat two oligonucleotides are linked to the polymeric delivery systemsprovided. Examples of this embodiment include oligonucleotides joined tothe polymer systems through their respective 3′-, 5′-terminal groups,e.g. 3′-bis oligonucleotide conjugates or 5′-bis oligonucleotideconjugates, or a conjugate formed by linking a first oligonucleotide viathe 3′- terminal to the 5′-terminal of a second oligonucleotide.Examples of such polymer conjugates are illustrated below as formulas(i), (ii), (iii) and (iv):

[0017] wherein all variables are as described above.

[0018] For purposes of the present invention, the term “residue” shallbe understood to mean that portion of a biologically active compoundi.e. an oligonucleotide, more specifically an antisense oligonucleotide,which remains after it has undergone a substitution reaction in whichthe prodrug carrier has been attached.

[0019] For purposes of the present invention, the term “polymericresidue” or “PEG residue” shall each be understood to mean that portionof the polymer or PEG which remains after it has undergone a reactionwith a modified oligonucleotide compound.

[0020] For purposes of the present invention, the term “alkyl” shall beunderstood to include straight, branched, substituted, e.g. halo-,alkoxy-, nitro-, C₁₋₁₂ alkyls, C₃₋₈ cycloalkyls or substitutedcycloalkyls, etc.

[0021] For purposes of the present invention, the term “substituted”shall be understood to include adding or replacing one or more atomscontained within a functional group or compound with one or moredifferent atoms.

[0022] For purposes of the present invention, substituted alkyls includecarboxyalkyls, aminoalkyls, dialkylaminos, hydroxyalkyls andmercaptoalkyls; substituted alkenyls include carboxyalkenyls,aminoalkenyls, dialkenylaminos, hydroxyalkenyls and mercaptoalkenyls;substituted alkynyls include carboxyalkynyls, aminoalkynyls,dialkynylaminos, hydroxyalkynyls and mercaptoalkynyls; substitutedcycloalkyls include moieties such as 4-chlorocyclohexyl; aryls includemoieties such as napthyl; substituted aryls include moieties such as3-bromo-phenyl; aralkyls include moieties such as toluyl; heteroalkylsinclude moieties such as ethylthiophene; substituted heteroalkylsinclude moieties such as 3-methoxy-thiophene; alkoxy includes moietiessuch as methoxy; and phenoxy includes moieties such as 3-nitrophenoxy.Halo- shall be understood to include fluoro, chloro, iodo and bromo.

[0023] The term “sufficient amounts” or “effective amounts” for purposesof the present invention shall mean an amount which achieves atherapeutic effect as such effect is understood by those of ordinaryskill in the art.

[0024] Some of the chief advantages of the present invention includenovel polymeric oligonucleotide prodrugs that demonstrate increasedstability and resistance to nuclease degradation, increased solubility,increased cell permeability and decreased toxicity.

[0025] Another advantage of the compounds of the invention is that avariety of polymeric prodrug platforms are releasably attached to themodified oligonucleotide compounds. This advantage allows for theartisan to design a drug conjugate that can be manipulated to includevarious moieties between the polymeric residue and the attachedoligonucleotide that can effect the rate of hydrolysis of the prodrug.The artisan thus has the ability to include substituents that allow formodulation of the rate of hydrolysis of the prodrug.

[0026] Methods of making and using the compounds, such as in methods oftreating cancers or malignancies, and conjugates described herein arealso provided. It is also contemplated that inventive polymericoligonucleotide prodrugs be administered together with (simultaneouslyand/or sequentially) any other suitable anticancer agent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 schematically illustrates methods of preparing thePEGylated oligonucleotides of compounds 3 and 5.

[0028]FIG. 2 schematically illustrates methods of preparing thePEGylated oligonucleotides of compounds 7 and 9.

[0029]FIG. 3 schematically illustrates methods of preparing thePEGylated oligonucleotides of compounds 11, 12 (SEQ ID NO: 1) and 14(SEQ ID NO: 1).

[0030]FIG. 4 schematically illustrates a method of preparing thePEGylated oligonucleotide of compound 16 (SEQ ID NO: 1).

[0031]FIG. 5 schematically illustrates methods of preparing thePEGylated oligonucleotides of compounds 17 (SEQ ID NO: 2), 18 (SEQ IDNO: 3) and 19 (SEQ ID NO: 4), from AS1 (SEQ ID NO:2), AS2 (SEQ ID NO:3)and AS3 (SEQ ID NO:4).

[0032]FIG. 6 schematically illustrates methods of preparing thePEGylated oligonucleotides of compounds 21 (SEQ ID NO: 1) and 22 (SEQ IDNO: 2).

[0033]FIG. 7 schematically illustrates methods of preparing thePEGylated oligonucleotides of compounds 24 (SEQ ID NO: 1) and 26 (SEQ IDNO: 1).

[0034]FIG. 8 schematically illustrates methods of preparing thePEGylated oligonucleotides of compounds 28 (SEQ ID NO: 1), 29 (SEQ IDNO: 2), 30 (SEQ ID NO: 3) and 31 (SEQ ID NO: 4), from AS1 (SEQ ID NO:2),AS2 (SEQ ID NO:3) and AS3 (SEQ ID NO:4).

[0035]FIG. 9 schematically illustrates methods of preparing thePEGylated oligonucleotides of compounds 33 (SEQ ID NO: 1) and 35 (SEQ IDNO: 1).

[0036]FIG. 10 illustrates the inhibitory effects of compound 14 andcompound 28 on PC3 cell growth. 0.4×10⁴ cells were seeded in 96-wellplates, treated either with complexes of compound 14 or compound 28 (400nM) and Lipofectin (15 μg/ml) for 24 hrs in Opti-MEM and then incomplete media without complexes. Cellular viability was determineddaily, and absorbances were measured at 570 nm. Data are presented asthe average ±standard deviation; n=4. Curves are as follows:

[0037] Control is marked by ♦ and a dotted curve;

[0038] Compound 28 at 400 nM is marked by  and a solid curve;

[0039] Compound 14 at 400 nM is marked by ▴ and a dashed curve;

[0040] Compound 28 at 200 nM is marked by  and a dashed curve;

[0041] Compound 14 at 200 nM is marked by ▪ and a dotted curve.

[0042]FIG. 11A provides a summary of ROS production (from flowcytometric analysis) by compound 14 and compound 28 oligonucleotides, bydetecting the oxidation of cell-permeable2′,7′-dihydrodichlorofluorescein diacetate to fluorescent2′,7′-dichlorofluorescein (DCF). PC3 cells were treated witholigonucleotides (400 nM)/Lipofectin (15 μg/ml) complexes for 24 hrs,and assayed after 3 days, as described. Fold increases in meanfluorescence channel were normalized against untreated cells.Experiments were done in triplicate and data are presented as mean±standard deviation (n=3).

[0043]FIG. 11B provides a summary of ROS production (from flowcytometric analysis) by compound 14 and compound 28 oligonucleotides, bydetecting the oxidation of hydroethidium (HE) to ethidium (E), whichsubsequently intercalates into DNA with fluorescence detectable by flowcytometry. PC3 cells were treated with oligonucleotides (400nM)/Lipofectin (15 μg/ml) complexes for 24 hrs, and assayed after 3days, as described. Fold increases in mean fluorescence channel werenormalized against untreated cells. Experiments were done in triplicateand data are presented as mean ±standard deviation (n=3).

[0044]FIG. 12 is a Western Blot results confirming inhibition of bcl-2protein expression by compound 14 in the presence of Lipofectin. PC3cells were treated with compound 14 oligonucleotide (200, 400 and 800nM) in the presence (+Lipo) and absence (−Lipo) of Lipofectin for 24 hrsin Opti-MEM, and then for a further 67 hrs in complete media. Proteinsamples (30-40 μg of protein/lane) were analyzed by Western blotting asdescribed in the Material and Methods, with tubulin used as a controlprotein species. “C” marks the control.

DETAILED DESCRIPTION OF THE INVENTION

[0045] Accordingly, the present invention provides for polymer-linkedoligonucleotide prodrugs useful having many practical uses, includinguses as diagnostic and analytic reagents, as research andinvestigational tools, both in vitro and in vivo, and as therapeuticagents. In order to more fully appreciate the scope of the presentinvention, the following terms are defined. The artisan will appreciatethat the terms, “nucleic acid” or “nucleotide” apply to deoxyribonucleicacid (“DNA”), ribonucleic acid, (“RNA) whether single-stranded ordouble-stranded, unless otherwise specified, and any chemicalmodifications thereof. An “oligonucleotide” is generally a relativelyshort polynucleotide, e.g., ranging in size from about 2 to about 200nucleotides, or more preferably from about 10 to about 30 nucleotides inlength. The oligonucleotides according to the invention are generallysynthetic nucleic acids, and are single stranded, unless otherwisespecified. The terms, “polynucleotide” and “polynucleic acid” may alsobe used synonymously herein.

[0046] Modifications to the oligonucleotides of the invention optionallyinclude, for example, the addition to or substitution of selectednucleotides with functional groups or moieties that permit covalentlinkage of an oligonucleotide to a desirable polymer, and/or theaddition or substitution of functional moieties that incorporateadditional charge, polarizability, hydrogen bonding, electrostaticinteraction, and functionality to an oligonucleotide. Such modificationsinclude, but are not limited to, 2′-position sugar modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodouracil, backbone modifications,methylations, base-pairing combinations such as the isobases isocytidineand isoguanidine, and analogous combinations. Oligonucleotidemodifications can also include 3′ and 5′ modifications such as capping.

[0047] The term “antisense,” as used herein, refers to nucleotidesequences which are complementary to a specific DNA or RNA sequence thatencodes a gene product or that encodes a control sequence. The term“antisense strand” is used in reference to a nucleic acid strand that iscomplementary to the “sense” strand. In the normal operation of cellularmetabolism, the sense strand of a DNA molecule is the strand thatencodes polypeptides and/or other gene products. The sense strand servesas a template for synthesis of an messenger RNA (“mRNA”) transcript (anantisense strand) which, in turn, directs synthesis of any encoded geneproduct. Antisense nucleic acid molecules may be produced by anyart-known methods, including synthesis by ligating the gene(s) ofinterest in a reverse orientation to a viral promoter which permits thesynthesis of a complementary strand. Once introduced into a cell, thistranscribed strand combines with natural sequences produced by the cellto form duplexes. These duplexes then block either the furthertranscription or translation. In this manner, mutant phenotypes may begenerated. The designations “negative” or (−) are also art-known torefer to the antisense strand, and “positive” or (+) are also art-knownto refer to the sense strand

[0048] For example, if it is intended to downregulate expression of anmRNA transcript in a cell or cells, the antisense oligonucleotide isintroduced into a cell. Once introduced into a cell, the antisenseoligonucleotide hybridizes to the corresponding mRNA sequence throughWatson-Crick binding, forming a heteroduplex. Once the duplex is formed,translation of the protein coded by the sequence of bound mRNA isinhibited. Thus, antisense oligonucleotides are also employed in the artas probes, e.g., hybridization probes, generally linked to a tag orlabel, as well as being used to provide precise downregulation of theexpression of specific cellular products or genetic regulatory elementsfor both investigational and therapeutic purposes.

[0049] For purposes of the present invention, the use of the singular orplural is not meant to be limiting of the numerical number of thereferenced item or object. Thus, the use of the singular to refer to acell, polymer or drug does not imply that only one cell is treated, onlyone molecule is prepared or employed, and/or only one drug is employed,and the use of the plural does not exclude application to a singlereferenced item, unless expressly stated.

[0050] For purposes of the present invention, the term “residue” shallbe understood to mean that portion of a biologically active compound,such as an oligonucleotide, which remains after it has undergone areaction in which the prodrug carrier portion has been attached bymodification of e.g., an available hydroxyl or amino group, to form, forexample, an ester or amide group, respectively.

[0051] A. Description of the Oligonucleotides

[0052] One of the features of the invention is the ability to provideimproved nucleotide or oligonucleotide polymer conjugates. The polymertransport systems described herein are not limited to a single speciesof oligonucleotide but, instead, are designed to work with a widevariety of such moieties, it being understood that the polymer transportsystems can attach to one or more of the 3′- or 5′- terminals, usuallyPO₄ or SO₄ groups of a nucleotide. The nucleotide sequences are depictedherein using conventional nomenclature, wherein the sequences are readfrom left to right, going from the 5′-terminus to the 3′-terminus (5′-→3′-).

[0053] X₁₋₃ represent the same or different nucleotide oroligonucleotide residue, which for purposes of the present inventioninclude oligodeoxynucleotide residues. More preferably, X₁₋₃ areindependently selected antisense oligonucleotide residues or antisenseoligodeoxynucleotide residues.

[0054] A non-limiting list of potential nucleotides which can be usedeither alone or as part of an oligonucleotide (10-1,000 nucleotides)include

[0055] wherein

[0056] M is O or S;

[0057] B₁ and B₂ are independently selected from the group consisting ofA (adenine), G (guanine), C (cytosine), T (thymine), U (uracil) andmodified bases, including those shown below and those known to those ofordinary skill;

[0058] R₁₀₀ and R₁₀₁ are independently selected from the groupconsisting of H, OR′ where R′ is H, a C₁₋₆ alkyl, substituted alkyl,nitro, halo, aryl, etc.

[0059] Some of the oligonucleotides and oligodeoxynucleotides useful inthe methods of the invention include, but are not limited to, thefollowing:

[0060] Oligonucleotides and oligodeoxynucleotides with naturalphosphorodiester backbone or phosphorothioate backbone or any othermodified backbone analogues;

[0061] LNA (Locked Nucleic Acid);

[0062] PNA (nucleic acid with peptide backbone);

[0063] tricyclo-DNA;

[0064] decoy ODN (double stranded oligonucleotide);

[0065] RNA (catalytic RNA sequence);

[0066] ribozymes;

[0067] spiegelmers (L-conformational oligonucleotides);

[0068] CpG oligomers, and the like, such as those disclosed at:

[0069] Tides 2002, Oligonucleotide and Peptide Technology Conferences,May 6-8, 2002, Las Vegas, Nev. and

[0070] Oligonucleotide & Peptide Technologies, 18th & 19th Nov. 2003,Hamburg, Germany, the contents of which are incorporated herein byreference.

[0071] Oligonucleotides according to the invention can also optionallyinclude any suitable art-known nucleotide analogs and derivatives,including those listed by Table 1, below. TABLE 1 RepresentativeNucleotide Analogs And Derivatives 4-acetylcytidine5-methoxyaminomethyl- 2-thiouridine 5-(carboxyhydroxymethyl)uridinebeta, D-mannosylqueuosine 2′-O-methylcytidine 5-methoxycarbonylmethyl-2-thiouridine 5-carboxymethylaminomethyl-2-5-methoxycarbonylmethyluridine thiouridine5-carboxymethylaminomethyluridine 5-methoxyuridine Dihydrouridine2-methylthio-N6- isopentenyladenosine 2′-O-methylpseudouridineN-((9-beta-D-ribofuranosyl-2- methylthiopurine- 6-yl)carbamoyl)threonineD-galactosylqueuosine N-((9-beta-D- ribofuranosylpurine-6-yl)N-methylcarbamoyl)threonine 2′-O-methylguanosine uridine-5-oxyacetic acid-methylester Inosine uridine-5-oxyacetic acid N6-isopentenyladenosinewybutoxosine 1-methyladenosine pseudouridine 1-methylpseudouridinequeuosine 1-methylguanosine 2-thiocytidine 1-methylinosine5-methyl-2-thiouridine 2,2-dimethylguanosine 2-thiouridine2-methyladenosine 4-thiouridine 2-methylguanosine 5-methyluridine3-methylcytidine N-((9-beta-D-ribofuranosylpurine-6-yl)-carbamoyl)threonine 5-methylcytidine 2′-O-methyl-5-methyluridineN6-methyladenosine 2′-O-methyluridine 7-methylguanosine wybutosine5-methylaminomethyluridine 3-(3-amino-3-carboxy- propyl)uridine

[0072] Preferably, the antisense oligonucleotide is one thatdownregulates a protein implicated in the resistance of tumor cells toanticancer therapeutics. For example, the protein BCL-2 inhibits therelease of Cytochrome-C and Apoptosis Initiating Factor frommitochondria and thus prevents apoptosis from occurring. Cancer cellsthat have high levels of BCL-2 are thus very resistant to bothchemotherapy or radiation therapy. U.S. Pat. No. 6,414,134, incorporatedby reference herein, describes antisense oligonucleotides thatdownregulate the protein Bcl-2 that is associated with resistance toanticancer therapy in a number of tumor cells, e.g., including prostatecancer cells, myeloma cells and other tumor cells. According to theabove-noted U.S. patent, the bcl-2 gene is believed to contribute to thepathogenesis of cancer primarily by prolonging tumor cell survivalrather than by accelerating cell division. U.S. Pat. No. 6,414,134generally describes antisense oligonucleotides of 17 to 35 bases inlength, that are complementary to bcl-2 mRNA, and that include a nucleicacid molecule having the sequence of TACCGCGTGC GACCCTC (SEQ ID NO: 5).These preferably include at least one phosphorothioate linkage.

[0073] Other art-known cellular proteins that are contemplated byvarious companies as targets for downregulation by antisenseoligonucleotides, for cancer therapy, are summarized by the followingtable. TABLE 2 Antisense Agent Target Protein Affinitak (ISIS 3521)PKC-alpha ISIS 112989 (OGX 011) Secretory Protein Clusterin ISIS 23722Survivin AP 12009 TGF-Beta2 GEM 231 Protein kinase A GEM 240 MDM2IGF-1R/AS ODN Insulin-like growth factor MG98 DNA methyltransferaseLErafAON C-raf-1 Ki-67 antisense Ki-67 oligonucleotide GTI-2040ribonucleotide reductase ISIS 2503 H-ras AP11014 TGF-Beta1

[0074] Antisense oligonucleotides suitable for use in downregulating theexpression of proteins related to cancer cell survival, such as bcl-2expression include oligonucleotides that are from about two to twohundred nucleotide codons; more preferably ten to forty codons; and mostpreferably about 17 to 20 codons. The oligonucleotides are preferablyselected from those oligonucleotides complementary to strategic sitesalong the pre-mRNA of bcl-2, such as the translation initiation site,donor and splicing sites, or sites for transportation or degradation.

[0075] Blocking translation at such strategic sites prevents formationof a functional bcl-2 gene product. It should be appreciated, however,that any combination or subcombination of anticode oligomers, includingoligonucleotides complementary or substantially complementary to thebcl-2 pre-mRNA or mRNA that inhibit cell proliferation is suitable foruse in the invention. For example, oligodeoxynucleotides complementaryto sequence portions of contiguous or non-contiguous stretches of thebcl-2 RNA may inhibit cell proliferation, and would thus be suitable foruse in methods of the invention.

[0076] Oligonucleotides suitable for downregulating bcl-2 expressionalso include oligonucleotides complementary or substantiallycomplementary to sequence portions flanking the strategic or other sitesalong the bcl-2 mRNA. The flanking sequence portions preferably rangefrom about two to about one hundred bases, upstream or downstream of thepreviously noted sites along the bcl-2 mRNA. These sites preferablyrange from about five to about twenty codons in length. It is alsopreferable that the oligonucleotides be complementary to a sequenceportion of the pre-mRNA or mRNA that is not commonly found in pre-mRNAor mRNA of other genes, in order to minimize homology of theoligonucleotides for pre-mRNA or mRNA coding strands from other genes.

[0077] A number of preferred antisense, or complementary,oligonucleotides for downregulating bcl-2 are listed as follows by Table3. TABLE 3 translation 3′ . . . CCCTTCCTACCGCGTGCGAC . . . 5′ (SEQ IDNO: 6) initiation antisense (TI-AS) bcl-2 5′ . . .CTTTTCCTCTGGGAAGGATGGCGCACGCTGGGAGA . . . 3′ (SEQ ID NO: 7) splice donor3′ . . . CCTCCGACCCATCCACGTAG . . . 5′ (SEQ ID NO: 8) antisense (SD-AS)bcl-2 5′ . . . ACGGGGTAC . . . GGAGGCTGGGTAGGTGCATCTGGT . . . 3′ (SEQ IDNO: 9) splice acceptor 3′ . . . GTTGACGTCCTACGGAAACA . . . 5′ (SEQ IDNO: 10) antisense (SA-AS) bcl-2 5′ . . .CCCCCAACTGCAGGATGCCTTTGTGGAACTGTACGG . . . 3′ (SEQ ID NO: 11)

[0078] It will be appreciated that antisense oligonucleotides can beemployed that comprise more or fewer substituent nucleotides, and/orthat extend further along the bcl-2 mRNA chain in either the 3′ or 5′direction relative to those listed by Table 3, supra.

[0079] Preferably, the antisense oligonucleotide employed in the prodrugof the invention has the same or substantially similar nucleotidesequence as does Genasense (a/k/a oblimersen sodium, produced by GentaInc., Berkeley Heights, N.J.). Genasense is an 18-mer phosphorothioateantisense oligonucleotide, TCTCCCAGCGTGCGCCAT (SEQ ID NO: 1), that iscomplementary to the first six codons of the initiating sequence of thehuman bcl-2 mRNA (human bcl-2 mRNA is art-known, and is described, e.g.,as SEQ ID NO: 19 in U.S. Pat. No. 6,414,134, incorporated by referenceherein). The U.S. Food and Drug Administration (FDA) has given GenasenseOrphan Drug status in August 2000, and has accepted a New DrugApplication (NDA) for Genasense in the treatment of cancer. The NDAproposes administering Genasense in combination with dacarbazine for thetreatment of patients with advanced melanoma who have not previouslyreceived chemotherapy. In addition, the FDA granted Priority Reviewstatus to the application, which targets an agency action on or beforeJun. 8, 2004. See also, Chi et al., 2001, Clinical Cancer Research Vol.7, 3920-3927, incorporated by reference herein, confirming activity ofGenasense in combination therapy of prostate cancer in early clinicaltrials. The prodrugs of the present invention have the same utility asthat recognized for the native (unmodified) 18-mer.

[0080] Genasense has been shown to downregulate the production of theBcl-2 protein and enhance a tumor cell's sensitivity to therapy andultimately, cause cell death. A number of studies have reportedpromising results in treatment of several cancers with Genasense incombination with anticancer agents. A phase I/II trial of Genasense incombination with dacarbazine in patients with melanoma has shownpromising activity and a Phase III multicenter trial is under way. Inaddition, Genasense, used in combination with mitoxantrone in patientswith hormone-refractory prostate cancer has shown promising results. Kimet al., 2001, Id.

[0081] The conjugation of antisense oligonucleotides, such as Genasense,to polymers exemplifies one preferred embodiment of the invention.

[0082] In an alternative embodiment, additional suitable antisenseoligonucleotides include: T-C-T-C-C-C-A-G-C-G-T-G-C-G-C-C-A-T; (compound13 - SEQ ID NO: 1) T-C-T-C-C-C-A-G-C-A-T-G-T-G-C-C-A-T; (compound 36 -SEQ ID NO: 2) A-T-C-C-T-A-A-G-C-G-T-G-C-G-C-C-T-T; (compound 37 - SEQ IDNO: 3) and T-C-T-C-C-C-A-G-X-G-T-G-X-G-C-C-A-T, (compound 38 - SEQ IDNO: 4)

[0083] as well as those found in the examples.

[0084] B. Formula (I)

[0085] In one preferred embodiment of the invention, there are providedoligonucleotide prodrugs of the formula (I):

[0086] wherein:

[0087] R₁ and R₂ are independently H or a polymer residue;

[0088] L₁ and L₄ are independently selected releasable linking moieties;

[0089] L₂ and L₃ are independently selected spacing groups;

[0090] X₁ is a nucleotide residue or an oligonucleotide residue; m, n, oand p are independently zero or a positive integer, provided that either(o+n) or (p+m)≧2.

[0091] The polymer transport system of the present invention is based inpart on the least one of R₁ and R₂ preferably being a polymeric residue,optionally having a capping group designated herein as A. Suitablecapping groups include, for example, OH, NH₂, SH, CO₂H, C₁₋₆ alkyls, aswell as oligonucleotide-containing groups such as

[0092] wherein X₂ and X₃ are either the same as X₁ or another nucleotideor oligonucleotide residue.

[0093] The preferred capping groups (II) and (III) allow compositions offormulas (i), (ii), (iii) and (iv) shown below to be formed:

[0094] wherein all variables are as previously described.

[0095] In another preferred embodiment of the invention, L₄ is areleasable linking moiety selected from among the formulas:

[0096] wherein:

[0097] Y₁₋₂₅ are independently selected from the group consisting of O,S or NR₉;

[0098] R₆₋₇, R₉₋₁₃, R₁₆₋₂₅, and R₂₇₋₄₁ are independently selected fromthe group consisting of hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls,C₃₋₈ cycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈ substituted cyloalkyls,aryls, substituted aryls, aralkyls, C₁₋₆ heteroalkyls, substituted C₁₋₆heteroalkyls, C₁₋₆ alkoxy, phenoxy and C₁₋₆ heteroalkoxy;

[0099] Ar is a moiety which forms a multi-substituted aromatichydrocarbon or a multi-substituted heterocyclic group;

[0100] L₅₋₁₂ are independently bifunctional spacers;

[0101] Z is selected from among moieties actively transported into atarget cell, hydrophobic moieties, bifunctional linking moieties andcombinations thereof;

[0102] c, h, k, l, r, s, v, w, v′, w′, c′, and h′ are independentlyselected positive integers;

[0103] a, e, g, j, t, z, a′, z′, e′ and g′ are independently either zeroor a positive integer; and

[0104] b, d, f, i, u, q, b′, d′ and f′ are independently zero or one.

[0105] In another preferred embodiment L₁ is a releasable linking moietyselected from among the formulas:

[0106] wherein

[0107] Y_(1′)-Y_(25′) are independently selected from the groupconsisting of O, S or NR₉;

[0108] R_(6′-7′), R_(9′-13′), R_(16′-25′), and R_(27′-41′)areindependently selected from the group consisting of hydrogen, C₁₋₆alkyls, C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆ substitutedalkyls, C₃₋₈ substituted cyloalkyls, aryls, substituted aryls, aralkyls,C₁₋₆ heteroalkyls, substituted C₁₋₆ heteroalkyls, C₁₋₆ alkoxy, phenoxyand C₁₋₆ heteroalkoxy; and

[0109] L_(5′-12′) are independently bifunctional spacers.

[0110] In some preferred embodiments of the invention, L₅₋₁₂ areindependently bifunctional spacers selected from among:

[0111] —(CH₂)₃—,

[0112] —C(O)NH(CH₂)₃—,

[0113] —NH(CH₂)₃—,

[0114] —(CR₅₅R₅₆)_(s)O(CR₅₇R₅₈)_(t)C(O)—

[0115] —NR₅₉(CH₂)(OCH₂CH₂)NH—

[0116] —(OCH₂CH₂)_(s)NH—

[0117] —O(CR₅₅R₅₆)_(s)NH—

[0118] —NR₅₉(CR₅₇R₅₈)_(t)C(O)NH(CR₅₅R₅₆)_(s)C(O)—

[0119] —O(CH₂)_(s)OC(O)—

[0120] —NR₅₉(CR₅₅R₅₆)_(s)C(O)—

[0121] —NR₅₉(CH₂)_(t)(OCH₂CH₂)_(s)NHC(O)—

[0122] —NR₅₉(OCH₂CH₂)_(s)OC(O)—

[0123] —O(CR₅₅R₅₆)_(s)NHC(O)—

[0124] —O(CR₅₅R₅₆)_(s)OC(O)—

[0125] (OCH₂CH₂)_(s)NHC(O)—

[0126] and L_(5′-12′) are independently bifunctional spacers selectedfrom among:

[0127] —(CH₂)₃—,

[0128] —(CH₂)₃NH—C(O),

[0129] —(CH₂)₃NH—,

[0130] —C(O)(CR_(57′)R_(58′))_(s′)O(CR_(55′)R_(56′))_(t′)

[0131] —NH(CH₂CH₂O)_(s′)(CH₂)_(t′)NR_(59′)—,

[0132] —NH(CH₂CH₂O)_(s′)—,

[0133] —NH(CR_(55′)R_(56′))_(s′)O—,

[0134] —C(O)(CR_(55′)R_(56′))_(s′)NHC(O)(CR_(57′)R_(58′))_(t′)NR_(59′)—,

[0135] —C(O)O(CH₂)_(s′)O—,

[0136] —C(O)(CR_(55′)R_(56′))_(s′)NR_(59′)—,

[0137] —C(O)NH(CH₂CH₂O)_(s′)(CH₂)_(t′)NR_(59′)—,

[0138] —C(O)O—(CH₂CH₂O)_(s′)NR_(59′)—,

[0139] —C(O)NH(CR_(55′)R_(56′))_(s′)O—,

[0140] —C(O)O(CR_(55′)R_(56′))_(s′)O—,

[0141] —C(O)NH(CH₂CH₂O)_(s′)—,

[0142] wherein:

[0143] R₅₅-R₅₉ and R_(55′-59′) are independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈cycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈ substituted cyloalkyls, arylssubstituted aryls, aralkyls, C₁₋₆ heteroalkyls, substituted C₁₋₆heteroalkyls, C₁₋₆ alkoxy, phenoxy and C₁₋₆ heteroalkoxy, and

[0144] R₆₀ and R_(60′) is selected from the group consisting ofhydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆substituted alkyls, C₃₋₈ substituted cyloalkyls, aryls substitutedaryls, aralkyls, C₁₋₆ heteroalkyls, substituted C₁₋₆ heteroalkyls, C₁₋₆alkoxy, phenoxy, C₁₋₆ heteroalkoxy, NO₂, haloalkyl and halogen; and

[0145] s′ and t′ are each a positive integer.

[0146] In another preferred embodiment of the invention, L₂ and L₃ areindependently spacing groups having about 1 to about 60 carbon atoms andfrom about 1 to about 10 heteroatoms. Preferably, L₂ and L₃ areindependently spacing groups having from about 2 to about 10 carbonatoms and from about 1 to about 6 heteroatoms. Most preferably, L₃ isselected from among:

[0147] -Q(CR₅₀R₅₁)_(q′)—,

[0148] -Q(CR₅₀R₅₁)q′O(CR₅₂R₅₃)r′

[0149] -Q(CH₂CH2O)_(q′)(CR₅₂R₅₃)_(r′)—,

[0150] -QCR₅₀R₅₁)_(q′)NHC(O)(CR₅₂R₅₃)_(′)—,

[0151]  and

[0152] -Q(CH₂)_(q′)—S—S—(CH₂)_(r′)—; and

[0153] most preferably, L₂ is selected from among:

[0154] —(CR_(50′)R_(51′))_(q′)Q′-,

[0155] —(CR_(52′)R_(53′))_(r′)O(CR_(50′)R_(51′))_(q′)Q′⁻

[0156] —(CR_(52′)R_(53′))_(r′)(OCH₂CH₂)Q′-,

[0157] —(CR_(52′)R_(53′))_(r′)C(O)NH(CR_(50′)R_(51′))_(q′)Q′-,

[0158]  and

[0159] —(CH₂)—S—S—(CH₂)_(q′)Q-

[0160] wherein.

[0161] Q and Q′ are independently selected from O, S or NH;

[0162] R₅₀₋₅₃ and R_(50′-53′), are independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈cycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈ substituted cyloalkyls, arylssubstituted aryls, aralkyls, C₁₋₆ heteroalkyls, substituted C₁₋₆heteroalkyls, C₁₋₆ alkoxy, phenoxy and C₁₋₆ heteroalkoxy;

[0163] R₅₄ and R_(54′) are independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈cycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈ substituted cyloalkyls, arylssubstituted aryls, aralkyls, C₁₋₆ heteroalkyls, substituted C₁₋₆heteroalkyls, C₁₋₆ alkoxy, phenoxy, C₁₋₆ heteroalkoxy, NO₂, haloalkyland halogen; and

[0164] q′ and r′ are each a positive integer.

[0165] With regard to the other variables which comprise the formulae ofthe present invention, the following are preferred:

[0166] Y₁₋₂₅ and Y_(1′-25′) are independently selected from the groupconsisting of O, S or NR₉;

[0167] R₆₋₇, R₉₋₁₃, R₁₆₋₂₅, R₂₇₋₄₁, and R_(6′-7′), R_(9′-13′),R_(16′-25′), and R_(27′-41′) are independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyls, C₃₋₈ cycloalkyls, aryls, aralkyls,and C₁₋₆ heteroalkyls;

[0168] c, h, k, l, r, s, v, w, v′, w′, c′, and h′ are one;

[0169] a, e, g, j, t, z, a′, z′, e′ and g′ are independently either zeroor one; and

[0170] b, d, f, i, u, q, b′, d′ and f′ are independently zero or one.

[0171] In yet another preferred embodiment of the invention there areprovided compounds of the formula (Ia):

[0172] wherein:

[0173] L₂ is a spacing group;

[0174] X₁ is a nucleotide or an oligonucleotide residue;

[0175] u′ is a positive integer; and

[0176] T is a branched polymer which is preferably selected from amongthose compounds described in commonly assigned PCT publication numbersWO02/065988 and WO02/066066, the disclosure of each being incorporatedherein by reference. Within these general formulae, the following arepreferred:

[0177] wherein R₆₁ is a polymer residue such as that defined for R₁ withthe understanding that the polymer can be bifunctional when R₆₁ is shownwith substitutions on both termini; and all other variables are asdescribed above.

[0178] For illustrative purposes, a non-limiting compound of formula(Ia) is:

[0179] wherein all variables are as described above.

[0180] Another aspect of formula (Ia) includes bifunctional compoundsthat are formed when the polymeric residue (R₆₁) includes both an alphaand an omega terminal linking group so that at least fouroligonucleotides are delivered. Examples of such polymer conjugates areillustrated below as formulas (vi) and (vii):

[0181] wherein all variables are as described above.

[0182] In another preferred embodiment of the invention, L₂ and L₃ areindependently spacing groups having about 1 to about 60 carbon atoms andfrom about 1 to about 10 hetero atoms. Preferably, L₂ and L₃ areindependently spacing groups having from about 2 to about 10 carbonatoms and from about 1 to about 6 heteroatoms. Most preferably, L₃ isselected from among:

[0183] -Q(CR₅₀R₅₁)_(q′)—,

[0184] -Q(CR₅₀R₅₁)q′O(CR₅₂R₅₃)r′

[0185] -Q(CH₂CH₂O)_(q′)(CR₅₂R₅₃)_(r′)—,

[0186] -QCR₅₀R₅₁)_(q′)NHC(O)(CR₅₂R₅₃)_(r′)—,

[0187]  and

[0188] -Q(CH₂)_(q′)—S—S—(CH₂)_(r′)—, and

[0189] most preferably, L₂ is selected from among:

[0190] —(CR_(50′)R_(51′))_(q′)Q′—,

[0191] —(CR_(52′)R_(53′))_(r′)O(CR_(50′)R_(51′))_(q′)Q′⁻,

[0192] —(CR_(52′)R_(53′))_(r′)(OCH₂CH₂)Q′-,

[0193] —(CR_(52′)R_(53′))_(r′)C(O)NH(CR_(50′)R_(51′))_(q′)Q′-,

[0194] and

[0195] —(CH₂)—S—S—(CH₂)_(q′)Q-

[0196] wherein.

[0197] Q and Q′ are independently selected from O, S or NH;

[0198] R₅₀₋₅₃ and R_(50′-53′)are independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈cycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈ substituted cyloalkyls, arylssubstituted aryls, aralkyls, C₁₋₆ heteroalkyls, substituted C₁₋₆heteroalkyls, C₁₋₆ alkoxy, phenoxy and C₁₋₆ heteroalkoxy;

[0199] R₅₄ and R_(54′) are independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈cycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈ substituted cyloalkyls, arylssubstituted aryls, aralkyls, C₁₋₆ heteroalkyls, substituted C₁₋₆heteroalkyls, C₁₋₆ alkoxy, phenoxy, C₁₋₆ heteroalkoxy, NO₂, haloalkyland halogen; and

[0200] q′ and r′ are each a positive integer.

[0201] With regard to the other variables which comprise the formulae ofthe present invention, the following are preferred:

[0202] Y₁₋₂₅ and Y_(1′-25′) are independently selected from the groupconsisting of O, S or NR₉;

[0203] R₆₋₇, R₉₋₁₃, R₁₆₋₂₅, R₂₇₋₄₁, and R_(6′-7′), R_(9′-13′),R_(16′-25′), and R_(27′-41′) are independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyls, C₃₋₈ cycloalkyls, aryls, aralkyls,and C₁₋₆ heteroalkyls;

[0204] c, h, k, l, r, s, v, w, v′, w′, c′, and h′ are one;

[0205] a, e, g, j, t, z, a′, z′, e′ and g′ are independently either zeroor one; and

[0206] b, d, f, i, u, q, b′, d′ and f′ are independently zero or one.

[0207] C. Description of the Ar Moiety

[0208] In certain aspects of the invention, it can be seen that the Armoiety is a moiety which when included in Formula (I) forms amulti-substituted aromatic hydrocarbon or a multi-substitutedheterocyclic group. A key feature is that the Ar moiety is aromatic innature. Generally, to be aromatic, the pi electrons must be sharedwithin a “cloud” both above and below the plane of a cyclic molecule.Furthermore, the number of pi electrons must satisfy the Huickel rule(4n+2). Those of ordinary skill will realize that a myriad of moietieswill satisfy the aromatic requirement of the moiety for formula (I) andthus are suitable for use herein.

[0209] Some particularly preferred aromatic groups include:

[0210] wherein R₆₂₋₆₇ are independently selected from the same groupwhich defines R₆.

[0211] Other preferred aromatic hydrocarbon moieties include, withoutlimitation

[0212] wherein E and E′ are independently CR₆₈ or NR₆₉; and J is O, S orNR₇₀ where R₆₈₋₇₀ are selected from the same group at that which definesR₆ or a cyano, nitro, carboxyl, acyl, substituted acyl or carboxyalkyl.Isomers of the five and six-membered rings are also contemplated as wellas benzo- and dibenzo-systems and their related congeners are alsocontemplated. It will also be appreciated by the artisan of ordinaryskill that aromatic rings can optionally be substituted withhetero-atoms such as O, S, NR₉, etc. so long as Huickel's rule isobeyed. Furthermore, the aromatic or heterocyclic structures mayoptionally be substituted with halogen(s) and/or side chains as thoseterms are commonly understood in the art.

[0213] D. Polyalkylene Oxides

[0214] Referring to Formula (I) it can be seen that R₁ and R₂ arepolymer moieties such as polyalkylene oxide. Suitable examples of suchpolymers include polyethylene glycols which are substantiallynon-antigenic. Also useful are polypropylene glycols, such as thosedescribed in commonly-assigned U.S. Pat. Nos. 5,643,575, 5,919,455 and6,113,906. Other PEG's useful in the methods of the invention aredescribed in Shearwater Polymers, Inc. catalog “Polyethylene Glycol andDerivatives 2001”. The disclosure of each is incorporated herein byreference. R₁ and R₂ are preferably PEG derivatives, e.g.—O—(CH₂CH₂O)_(x)— Within this aspect, R₁₋₂ are independently selectedfrom among:

[0215] J-O—(CH₂CH₂O)_(n′)—

[0216] J-O—(CH₂CH₂O)_(n′)—CH₂C(O)—O—,

[0217] J-O—(CH₂CH₂O)_(n′)—CH₂CH₂ NR₄₈—,

[0218] J-O—(CH₂CH₂O)_(n′)—CH₂CH₂ SH,

[0219] —O—C(O)CH₂—O—(CH₂CH₂O)_(n′)—CH₂C(O)—O—,

[0220] —NR₄₈CH₂CH₂—O—(CH₂CH₂O)_(n′)—CH₂CH₂ NR₄₈—,

[0221] —SHCH₂CH₂—O—(CH₂CH₂O)_(n′)—CH₂CH₂ SH—,

[0222] wherein

[0223] n′ is the degree of polymerization selected so that the weightaverage molecular weight is at least about 2,000 Da to about 136,000 Da;

[0224] R₄₈ is selected from the group consisting of hydrogen, C₁₋₆alkyls, C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆ substitutedalkyls, C₃₋₈ substituted cyloalkyls, aryls substituted aryls, aralkyls,C₁₋₆ heteroalkyls, substituted C₁₋₆ heteroalkyls, C₁₋₆ alkoxy, phenoxyand C₁₋₆ heteroalkoxy; and

[0225] J is a capping group such as methyl or a complementary linkinggroup which allows a bifunctional polymer to be provided.

[0226] Although PAO's and PEG's can vary substantially in weight averagemolecular weight, preferably, R₁ and R₂ independently have a weightaverage molecular weight of from about 2,000 Da to about 136,000 Da inmost aspects of the invention. More preferably, R₁ and R₂ independentlyhave a weight average molecular weight of from about 3,000 Da to about100,000 Da, with a weight average molecular weight of from about 5,000Da to about 40,000 Da being most preferred.

[0227] The polymeric substances included herein are preferablywater-soluble at room temperature. A non-limiting list of such polymersinclude polyalkylene oxide homopolymers such as polyethylene glycol(PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymersthereof and block copolymers thereof, provided that the water solubilityof the block copolymers is maintained.

[0228] E Synthesis of Oligonucleotide Polymer Conjugates

[0229] Generally the prodrugs are prepared by

[0230] a) reacting a compound of the formula:

R₂-L₄-leaving group

[0231] with a compound of the formula:

H-L₃-X₁

[0232] under conditions sufficient to form a prodrug of the formula

R₂-L₄-L₃-X₁,

[0233] wherein:

[0234] R₂ is a polymer residue;

[0235] L₄ is a releasable linking moiety;

[0236] L₃ is a spacing group;

[0237] X₁ is a nucleotide or an oligonucleotide residue.

[0238] Within this aspect of the invention, it is preferred to employactivated polymers which already contain the releasable linkers attachedthereto. A non-limiting list of suitable combinations include thereleasable PEG-based transport systems described in commonly-assignedU.S. Pat. Nos. 6,624,142, 6,303,569, 5,965,119, 6,566,506, 5,965,119,6,303,569, 6,624,142, and 6,180,095. the contents of each areincorporated herein by reference.

[0239] Specific examples include but are not limited to,

[0240] it being understood of course that the molecular weight of thepolymer portion can be varied according to the needs of the artisan.

[0241] The polymer releasable linker shown above is then reacted with amodified oligomer under conditions sufficient to allow the conjugate tobe formed.

[0242] Any of the nucleotides or oligonucleotides described above can befunctionalized on one of the 5′- or 3′-terminal phosphate orphosphorothioate using routine techniques such as the phosphoramiditemethods to attach a desired alkyl-amino or other group onto the terminalphosphate. For example, a blocked (Fmoc) amino alkyl is attached, theresultant compound is oxidized, deprotected and purified.

[0243] Synthesis of specific oligonucleotide polymer conjugates orprodrugs is set forth in the Examples. Alternatively, the prodrugs canbe prepared by:

[0244] 1) reacting an activated PEG polymer with a protectedbifunctional releasable linking group under suitable coupling conditionsto form a first intermediate,

[0245] 2) deprotecting and activating the intermediate of step 1) with asuitable activating group such as NHS ester, and

[0246] 3) reacting the activated intermediate of step 2) with a modifiedoligonucleotide in a PBS buffered system to obtain the desiredoligonucleotide polymer prodrug.

[0247] A non-limiting list of activated polymers includebis-succinimidyl carbonate activated PEG (SC-PEG),bis-thiazolidine-2-thione activated PEG (T-PEG), N-hydroxyphthalamidylcarbonate activated PEG (BSC-PEG),(see commonly assigned U.S. Ser. No.09/823,296, the disclosure of which is incorporated herein byreference), succinimidyl succinate activated PEG (SS-PEG) andmono-activated PEG's such as those found in, for example, in theaforementioned 2001 Shearwater Catalog.

[0248] Conjugation of the activated PEG polymer to the protectedbifunctional releasable linking group can be carried out in the presenceof a coupling agent. A non-limiting list of suitable coupling agentsinclude 1,3-diisopropylcarbodiimide (DIPC), any suitable dialkylcarbodiimide, 2-halo-1-alkyl-pyridinium halides (Mukaiyama reagents),1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC), propane phosphonicacid cyclic anhydride (PPACA) and phenyl dichlorophosphates, etc. whichare available, for example from commercial sources such as Sigma-AldrichChemical, or synthesized using known techniques.

[0249] Preferably the substituents are reacted in an inert solvent suchas tetrahydrofuran (THF), acetonitrile (CH₃CN), methylene chloride(DCM), chloroform (CHCl₃), dimethyl formamide (DMF) or mixtures thereof,and at a temperature from 0° C. up to about 22° C. (room temperature).Conjugation of the modified oligonucleotide to the PEG-releasable linkercan be carried out in a PBS buffered system in the pH range of about7.4-8.5. The artisan of course will appreciate that synthesis of theprodrugs described herein, will also include the use of commonly foundlaboratory conditions, i.e. solvents, temperature, coupling agents etc.such as those described in the examples.

[0250] Regardless of the synthesis selected, some of the preferredcompounds which result from the synthetic techniques described hereininclude:

[0251] represent an oligonucleotide and point of terminal phosphatemodification and mPEG is CH₃—O—(CH₂—CH₂—O)_(x)—; wherein x is a positiveinteger selected from about 10 to about 2300.

[0252] More preferred compounds of the invention include:

[0253] G. Methods of Treatment

[0254] Another aspect of the present invention provides methods oftreatment for various medical conditions in mammals. The methods includeadministering to the mammal in need of such treatment, an effectiveamount of an oligonucleotide prodrug, which has been prepared asdescribed herein. The compositions are useful for, among other things,treating neoplastic disease, reducing tumor burden, preventingmetastasis of neoplasms and preventing recurrences of tumor/neoplasticgrowths, liver diseases, viral diseases such as, HIV, in mammals. Theprodrugs of the present invention can be used for whatever indicationthe native olignucleotide or antisense oligonucleotides are used for,i.e. cancer therapy, etc. Simply by way of example, the inventiveprodrugs are contemplated to be employed in the treatment of multiplemyeloma, chronic lymphocytic leukemia, non-small cell lung cancer, smallcell lung cancer, prostate cancer and other tumors or cancers toonumerous to mention.

[0255] The amount of the prodrug administered will depend upon theparent molecule included therein an condition being treated. Generally,the amount of prodrug used in the treatment methods is that amount whicheffectively achieves the desired therapeutic result in mammals.Naturally, the dosages of the various prodrug compounds will varysomewhat depending upon the parent compound, rate of in vivo hydrolysis,molecular weight of the polymer, etc. The range set forth above isillustrative and those skilled in the art will determine the optimaldosing of the prodrug selected based on clinical experience and thetreatment indication. Actual dosages will be apparent to the artisanwithout undue experimentation.

[0256] The prodrugs of the present invention can be included in one ormore suitable pharmaceutical compositions for administration to mammals.The pharmaceutical compositions may be in the form of a solution,suspension, tablet, capsule or the like, prepared according to methodswell known in the art. It is also contemplated that administration ofsuch compositions may be by the oral and/or parenteral routes dependingupon the needs of the artisan. A solution and/or suspension of thecomposition may be utilized, for example, as a carrier vehicle forinjection or infiltration of the composition by any art known methods,e.g., by intravenous, intramuscular, subdermal injection and the like.

[0257] Such administration may also be by infusion into a body space orcavity, as well as by inhalation and/or intranasal routes. In preferredaspects of the invention, however, the prodrugs are parenterallyadministered to mammals in need thereof.

[0258] It is also contemplated that the prodrugs of the invention beadministered in combination (e.g., simultaneously and/or sequentially)with other art-known anticancer agents. Suitable anticancer agentsinclude, simply by way of example: (Paclitaxel; Bristol Myers Squibb);Camptosar® (Irinotecan; Pfizer.); Gleevec® (Imatiinib Mesylate;Novartis); Rituxan® (Rituximab; Genentech/IDEC); Fludara® (Fludarabine;Berlex Labs); Cytoxan(® (cyclophosphamide; Bristol Myers Squibb);Taxotere® (Docetaxel; Aventis Pharmaceuticals); Mylotarg® (Gemtuzumabozogamicin; Wyeth-Ayerst); Cytosine arabinoside and/or dexamethasone, toname but a few such agents.

EXAMPLES

[0259] The following examples serve to provide further appreciation ofthe invention but are not meant in any way to restrict the effectivescope of the invention. The underlined and bold-faced numbers recited inthe Examples correspond to those shown in the Figures. In each of thefigures, the sugar moiety and phosphate backbone are represented as:

[0260] The MPEG designation shall be understood to represent

[0261] General Procedures. All the conjugation reactions between PEGlinkers and oligonucleotides were carried out in PBS buffer systems atroom temperature. Extraction with organic solvents in general removedthe un-reacted oligonucleotides, further anion-exchange chromatographyseparated PEG-Oligo conjugates from non-reacted excess_PEG linkers togive pure products.

[0262] HPLC methods. The reaction mixtures and the purity ofintermediates and final products were monitored by a Beckman CoulterSystem Gold® HPLC instrument employing a ZORBAX® 300 SB C-8 reversedphase column (150×4.6 mm) or a Phenomenex Jupiter® 300A C18 reversedphase column (150×4.6 mm) with a multiwavelength UV detector, using agradient of 30-90% of acetonitrile in 0.5 % trifluoroacetic acid (TFA)and 25-35% acetonitrile in 50 mM TEAA buffer with 4 mM TBACl at a flowrate of 1 mL/min. The anion exchange chromatography was run on Bio-Cad700E Perfusion Chromatography Workstation from Applied Biosystems usingeither Poros 50HQ strong anion exchange resin from Applied Biosystems orDEAE Sepharose fast flow weak anion exchange resin from AmershamBiosciences packed in an AP-Empty glass column from Waters. Desaltingwas achieved by using HiPrep 26/10 or PD-10 desalting columns fromAmersham Biosciences.

Example 1

[0263] Compound 3. A solution of compound 1 (440 mg, 0.036 mmol) and 2(5 mg, 3.6 μmol) in PBS buffer (10 mL, pH 7.4) was stirred at roomtemperature for 12 hrs. The reaction solution was extracted withmethylene chloride (DCM, 3×10 mL) and the combined organic layers weredried (MgSO₄), filtered, and the solvent evaporated under reducedpressure. The residue was dissolved in double distilled water (1.5 mLper 100 mg) and loaded on a HQ/10 Poros strong anion exchange column (10mm×60 mm, bed volume ˜6 mL). The un-reacted PEG linkers were eluted withwater (3˜4 column volumes) and the product then eluted with 0.2 MNH₄HCO₃ solution (˜2 column volumes). The fractions containing pureproduct were pooled and lyophilized to yield pure 3 (19 mg, 1.44 mmol,40%).

Examples 2-6

[0264] Compounds 5, 7, 9, 11, and 12 were made and purified in thesimilar way as 3 in the yields ranging from 30% to 50%.

Example 7

[0265] Compound 14. To a solution of compound 13 (10 mg, 1.7 μmol) inPBS buffer (5 mL, pH 7.4) was added 10 (175 mg, 85 μmol) in fiveequivalent portions every hour and stirred at room temperature for 12hrs. The reaction solution was extracted with DCM (3×10 mL) and brine(10 mL), and the combined organic layers were dried (MgSO₄), filtered,and the solvent evaporated under reduced pressure. The residue wasdissolved in double distilled water (1.5 mL) and loaded on a DEAE fastflow, weak anion exchange column (10 mm×60 mm, bed volume ˜6 mL) whichwas pre-equilibrated with 20 mM tris-HCl buffer, pH 7.4. The unreactedPEG linkers were eluted with water (3 to 4 column volumes) and theproduct then eluted with a gradient of 0 to 100% 1 M NaCl in 20 mMTris-HCl buffer 7.4 in 10 min, followed by 100% 1M NaCl for 10 min at aflow rate of 3 mL/min. The fractions containing pure product were pooledand desalted on a PD-10 desalting column with 0.2 M NH₄HCO₃ solution (˜2column volumes) and the resulting solution was lyophilized to yield pure14 (25 mg, 0.95 μmol, 57%).

Example 8

[0266] Compound 16 was made and purified the same way as 14, with a 60%yield.

Example 9

[0267] Compound 17. To a solution of AS1 (5 mg, 0.85 μmol) in phosphatebuffer (2 mL, pH 7.8) was added 10 (175 mg, 0.085 mmol) which wasdivided into five equivalent portions in 2 hrs and the resultingsolution stirred at room temperature for another 2 hrs. The reactionsolution was extracted with DCM (3×6 mL) and brine (5 mL), and thecombined organic layers were dried (MgSO₄), filtered, and the solventevaporated under reduced pressure. The residue was dissolved in doubledistilled water (5 mL) and loaded on a DEAE fast flow, weak anionexchange column (10 mm×60 mm, bed volume ˜6 mL) which waspre-equilibrated with 20 mM tris-HCl buffer, pH 7.4. The unreacted PEGlinkers were eluted with water (3 to 4 column volumes) and the productthen eluted with a gradient of 0 to 100% of 1 M NaCl in 20 mM Tris-HClbuffer, pH 7.4, for 10 minutes followed by 100% 1M NaCl at a flow rateof 3 mL/min for 10 minutes. The fractions containing pure product werepooled and desalted on a PD-10 desalting column and the resultingsolution was lyophilized to yield pure 17 (15 mg, 0.57 μmol, 67%).

Examples 10-11

[0268] Compounds 18 and 19 were made and purified the same way as 17with yields of 67% for the final products using AS2 and AS3 in place ofAS1.

Examples 12-15

[0269] Compound 21 was made and purified the same way as 14 by replacing12 with 20 with a yield of 90%.

[0270] Compound 22 was made and purified in the same way as 14 byreplacing 12 with 20 with a yield of 65%.

[0271] Compound 24 was made and purified in the same way as 14 byreplacing 12 with 23 and, for desalting, water (˜2 column volumes) wasused to elute the product instead of 0.2 M NH₄HCO₃ solution. The finalwas 30%.

[0272] Compound 26 was made and purified in the same way as 24 byreplacing 23 with 25. The yield was 30%.

Example 16

[0273] Compound 27. To a solution of 13 (10 mg, 1.7 μmol) in phosphatebuffer (5 mL, pH 8.5) was added 27 (180 mg, 0.084 mmol) which wasdivided into ten equivalent portions, and the resulting solution stirredat room temperature for 4 days. The reaction solution was extracted withDCM (3×10 mL) and brine (10 mL), and the combined organic layers weredried (MgSO₄), filtered, and the solvent evaporated under reducedpressure. The residue was dissolved in double distilled water (1.5 mL)and loaded on a DEAE fast flow, weak anion exchange column (10 mm×60 mm,bed volume ˜6 mL) which was pre-equilibrated with 20 mM tris-HCl buffer,pH 7.4. The un-reacted PEG linkers were eluted with water (3 to 4 columnvolumes) and the product then eluted with a gradient of 0 to 100% 1 MNaCl in 20 mM Tris-HCl buffer, pH 7.4, for 10 min, followed by 100% 1MNaCl at a flow rate of 3 mL/min for 10 minutes. The fractions containingpure product were pooled and desalted on a PD-10 desalting column andthe resulting solution was lyophilized to yield 14 (105 mg, 0.0102 mmol,60%). The purity of the product was determined by HPLC.

Examples 17-21

[0274] Compounds 29, 30 and 31 were made and purified the same way as28, except that 13 was replaced by AS1, AS2 and AS3, respectively. Ayield of 65% was obtained for each of the final products.

[0275] Compound 33 was made and purified the same way as 14 with 32replacing 12 resulting in a yield of 76%.

[0276] Compound 35 was made and purified the same way as 14 except thatactivated PEG 34 was used in place of 10. The final yield was 30%.

Biological Data

[0277] Some in vitro properties of PEG-Oligo conjugates are summarizedbelow: TABLE 4 In vitro properties of PEG-Oligo conjugates t_(1/2)t_(1/2) (Rat t_(1/2) t_(1/2) t_(1/2) Compound (buffer) Plasma) (PE I)(PE II) (NucleaseP1) 3 >>24 h  0.7 h <5 min >24 h <5 min 7 >>24 h  0.5 h<5 min >24 h <5 min 5 >>24 h  0.5 h <5 min >24 h <5 min 9 >>24 h  3.7 h<5 min 36 0.3 h 11 >>24 h  2.3 h <5 min 63 0.3 h 12 >>24 h 10.6 h <5 min1.7 <5 min 14 >>24 h 14.7 h <5 min 38.9 0.3 h 16 >>24 h 13.8 h <5 min42.7 0.3 h 21 >>24 h  6.5 h >24 h >24 h <5 min 24 >>24 h  11. h 18.7 h 36.8 h  <5 min 28 >>24 h  >48 h >24 h >48 h <5 min 33 >>24 h  >48 h >24h >48 h <5 min 35 >>24 h  4.1 h  5.6 h >48 h <5 min

[0278] Pharmacokinetics of PEG-Oligo Conjugate in ICR Mice

[0279] General Procedures.

[0280] 1) Animal Husbandry: Mice were housed 6 per cage, in breederboxes. Cages were sized in accordance with the “Guide for the Care andUse of Laboratory Animals of the Institute of Laboratory AnimalResource”, National Research Council.

[0281] 2) Diet: The mice had access to tap water and were fedcommercially available lab chow ad libitum.

[0282] 3) Compound Preparation: Compound 13 was dissolved in 4.0 mL ofsaline and compound 14 was dissolved in 4.1 mL of saline.

[0283] 4) Administration Site: Compound 13 and 14 were administered as asingle dose (Day 1) via the tail vein.

[0284] Experimental Design

[0285] Sixty (60) mice were assigned, dosed and bled according to thefollowing design, shown by Table 5, below. TABLE 5 Dose Dose* Vol Grp TxN (mg/kg) (mg/kg) Inj Time Points Bled (h) (μl) 1 14 3 120 4 iv 0.031000 2 14 3 120 4 iv 0.25 1000 3 14 3 120 4 iv 0.5 1000 4 14 3 120 4 iv1 1000 5 14 3 120 4 iv 3 1000 6 14 3 120 4 iv 6 1000 7 14 3 120 4 iv 241000 8 14 3 120 4 iv 48 1000 9 14 3 120 4 iv 72 1000 10 14 3 120 4 iv 961000 11 13 3 4 4 iv 0.03 1000 12 13 3 4 4 iv 0.25 1000 13 13 3 4 4 iv0.5 1000 14 13 3 4 4 iv 1 1000 15 13 3 4 4 iv 3 1000 16 13 3 4 4 iv 61000 17 13 3 4 4 iv 24 1000 18 13 3 4 4 iv 48 1000 19 13 3 4 4 iv 721000 20 13 3 4 4 iv 96 1000

[0286] Three (3) untreated mice were bled via cardiac puncture into EDTAcontaining tubes for the collection of untreated control plasma.

[0287] The mice were injected intravenously with 100 μL per mouse withnative compound 13 and 14. Following sedation with 0.09% Avertin, themice were terminally bled ˜1000 μL by cardiac puncture. Blood wascollected into EDTA containing vials. The plasma was collected followingcentrifugation of the blood and immediately frozen at −80° C. on dryice.

[0288] Clinical Examinations:

[0289] The mice were examined visually once daily following infusion ofthe test article, for mortality and signs of reaction to treatment. Anydeath and clinical signs was recorded. Body weights were measured prioron the day of injection only.

[0290] The pharmacokinetic studies for compounds 28 and 33 were done ina similar fashion.

[0291] Experimental Results

[0292] The pharmacokinetic results are summarized in the following Table6, below. TABLE 6 In vivo properties of PEG-Oligo conjugates Cmax PlasmaHalf- AUC Compound (μg/mL) life (hr) (hr · μg/mL) 13 14.9 0.19 4.1 1454.8 0.66 51.8 28 491.2 1.05 730.6 33 556.7 0.25 191.0

Examples 22-25

[0293] Confirmation of In Vitro Activity of Antisense PEG Conjugates

[0294] Bcl-2 protein has been shown to have significant anti-apoptoticactivity in prostate cancer cells. Downregulation of bcl-2 protein inprostate cancer cells is confirmed by cell death, and induction of celldeath by bcl-2 antisense PEG conjugates was employed to confirm thesuccessful intra-cellular delivery of the antisense oligonucleotides.

[0295] Materials And Methods For Examples 22-25

[0296] The tested compounds are listed by Table 6, below. TABLE 7Compound Description 14 5′ G aromatic releasable 28 5′- permanent 33 5′G-->A aromatic releasable 24 24k-mPEG-BCN3-5′ aliphatic releasable 3520k-mPEG-RNL9 5′ releasable (intermediate t_(1/2) in rats)

[0297] These were prepared as described supra.

[0298] Cell Culture

[0299] Mycoplasma-free PC3 cells were obtained from the American TypeCulture Collection (Rockville, Md.) were grown in Roswell Park MemorialInstitute media (“RPMI”) (Invitrogen, Grand Island, N.Y.) plus 10% fetalbovine serum (“FBS”), containing 10% (v/v) heat inactivated (56° C.) FBSsupplemented with 1% non-essential amino acids, 1% pyruvate, 25 mM HEPES(N-2-Hydroxyehtylpiperazine-N′-2-ethanesulfonic acid) buffer, 100 U/mlpenicillin G sodium and 100 μg/ml streptomycin sulfate. Stock cultureswere maintained at 37° C. in a humidified 5% CO₂ incubator.

[0300] Reagents

[0301] FBS and Lipofectin (liposome formulation of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride) werepurchased from Invitrogen (Grand Island, N.Y.). The anti-bcl-2monoclonal antibody was from Dako (Carpinteria, Calif.). Theanti-α-tubulin monoclonal antibody and3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (“MTT”)were purchased from Sigma-Aldrich (St. Louis, Mo.). Phosphorothioateoligonucleotides were synthesized and purified via standard procedures.

[0302] Oligonucleotide Transfection

[0303] Cells were seeded the day before the experiment in 6-well platesat a density of 25×104 cells per well, to be 60-70% confluent on the dayof the experiment. All transfections were performed in Opti-MEM mediumin the absence of FBS and antibiotics as per the manufacturer'sinstructions. The appropriate quantities of reagents were diluted in 100μl of Opti-MEM medium to give a final concentration of Lipofectin andoligonucleotide. The solutions were mixed gently and preincubated atroom temperature for 30 minutes to allow complexes to form. Then, 800 μlof Opti-MEM was added, the solution mixed, and overlaid on the cellsthat had been pre-washed with Opti-MEM. The incubation time foroligonucleotide/Lipofectin complexes in Opti-MEM was 24 hrs, followed byincubation in complete media containing 10% FBS. The total incubationtime before cell lysis and protein isolation was usually 72 h at 37° C.

[0304] Western Blot Analysis

[0305] Cells treated with oligonucleotide-lipid complexes were washed inPBS and then extracted in lysis buffer [50 mM Tris-HCl pH 7.4, 1% NP-40,0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 50 μg/ml Pefabloc SC,15 μg/ml aprotinin, leupeptin, chymostatin, pepstatin A, 1 mM Na3VO4, 1mM NaF] at 4° C. for 1 h. Cell debris was removed by centrifugation at14 000 g for 20 rain at 4° C. Protein concentrations were determinedusing the Bio-Rad protein assay system (Bio-Rad Laboratories, Richmond,Calif.). Aliquots of cell extracts, containing 25-40 μg of protein, wereresolved by SDS-PAGE, and then transferred to Hybond ECL filter paper(Amersham, Arlington Heights, Ill.), and the filters incubated at roomtemperature for 1-2 hr in 5% BSA in PBS containing 0.5% Tween-20. Thefilters were then probed with 1:500 dilutions of the anti bcl-2 antibodyin 5% BSA in PBS containing 0.5% Tween-20 at 4° C. overnight. Afterwashing in PBS containing 0.5% Tween-20, the filters were incubated for1 hour at room temperature in 5% milk in PBS containing 0.5% Tween witha 1:3,000 dilution of a peroxidase-conjugated secondary antibody(Amersham). After washing (3×10 min), electrochem-iluminescense (“ECL”)was performed according to the manufacturer's instructions.

[0306] Determination of Rate of Cell Proliferation

[0307] The effect on cellular viability of PEG conjugates was determinedby MTT assay. Briefly, 15-20×10⁴ cells were seeded in 6-well plates andallowed to attach overnight. Cells were then treated with theappropriate concentrations of oligonucleotide complexed to Lipofectinfor 24 hrs at 37° C., followed by incubation in complete media (100 μl)containing 10% FBS. Cell viability was determined daily. Ten μl of 5mg/ml MTT in PBS was added to each well, followed by incubation for 4 hat 37° C. Then, 100 μl of 10% SDS in 0.04 M HCl was added to each well,followed by incubation for overnight at 37° C. to dissolve the formazancrystals. Absorbance was determined at 570 mn with a Benchmark plusMicroplate spectrophotometer (Bio Rad, Hercules, Calif.). Experimentswere performed in 6 replicates, and data are presented as the average+/− S.D.

[0308] Quantitation of Intracellular ROS Levels

[0309] 2′,7′-dichlorodihydrofiuorescein diacetate (H₂DCF-DA) anddihydroethidium (HE) were used to determined reactive oxygen species(“ROS”) and superoxide levels. Both dyes are nonfluorescent and canfreely diffuse into cells. When HE is oxidized to ethidium (E), itintercalates into cellular DNA and fluoresces. Oxidation of H₂DCF-DAyields 2′-7′ dichlorofluorescein (DCF), which also fluoresces, and bothcan be detected by flow cytometry. Cells were harvested bytrypsinization, washed with PBS and stained with 50 μM H₂DCF-DA or 50 μMHE in phenol red-free DMEM for 2 h at 37° C. The mean fluorescencechannel numbers of DCF and E were analyzed by flow cytometry in the FL-1and FL-2 channels respectively. A minimum of 10,000 cells were acquiredfor each sample and data were analyzed using CELLQuest software (BectonDickinson). Histograms were plotted on a logarithmic scale.

Example 22

[0310] Inhibition Of Bcl-2 Protein Expression

[0311] Three PEG oligonucleotides (compounds 14, 28 and 33) targeted tobcl-2 expression were transfected into PC3 cells and their capacity toinhibit bcl-2 protein expression was evaluated by Western blotting.

[0312] Effects of Lipofectin

[0313] Initially, the degree of inhibition of bcl-2 protein expressionin PC3 cells induced by compound 14 was determined in the presence andabsence of Lipofectin. PC3 cells were treated with compound 14 (at 200,400 and 800 nM) in the presence or absence of Lipofectin for 24 hrs inOpti-MEM, and then for a further 67 hrs in complete media. Proteinsamples (30-40 μg of protein/lane) were analyzed by Western blotting asdescribed in the Material and Methods, with tubulin used as a controlprotein species. The % inhibition vs. control, untreated cells wasdetermined by laser-scanning densitometry. The Western Blot results areshown by FIG. 12.

[0314] The expression of α-tubulin and PKCα was unchanged, confirmingthat Lipofectin is helpful to obtaining penetration of compound 14 intoPC-3 cells, and confirming that only bcl-2 protein expression wasdownregulated by compound 14.

[0315] Further investigation demonstrated that compounds 14 and 28 werethe most active at 400 nM. PC3 cells were treated with complexes ofcompound 14, compound 28, and compound 23 at 400, 800 and I000 nM andLipofectin for 24 hrs in Opti-MEM, and then for a further 67 hrs incomplete media. Protein samples (30-40 μg of protein/lane) were analyzedby Western blotting as described in the Material and Methods, withtubulin used as a control protein species. The % inhibition vs. control,untreated cells was determined by laser-scanning densitometry.

[0316] Compound 14 produced 86% downregulation and compound 28 produced78% downregulation.

Example 23

[0317] Dose-Dependent Analysis of bcl-2 Protein Expression by PEGOligonucleotides

[0318] To further confirm the inhibitory effect of compounds 14 and 28on bcl-2 protein expression, PC3 cells were treated with increasingconcentrations (25, 50, 100, 200 and 400 nM) of compound 14, compound28, and compound 13 as a positive control, complexed to Lipofectin, for24 hrs in Opti-MEM, and then for a further 67 hrs in complete media.Protein samples (30-40 μg of protein/lane) were analyzed by Westernblotting as described in the Material and Methods section, supra.

[0319] A concentration-dependent inhibition of bcl-2 protein expressionwas observed by Western Blot for compounds 14 and 28, relative tocontrols. About 1-2% inhibition was observed at 50 nM, increasing to 99%and 75% at a concentration of 400 nM. For compound 24, essentially noinhibition was observed at 50 nM, but the inhibition increased to 77% ata concentration of 400 nM. Transfection with compound 13 was used as apositive control. The expression of α-tubulin was not inhibited by anyoligonucleotide.

[0320] The above experiment was repeated with compound 24 as a control.PC3 cells were treated with increased concentrations of compounds 35 and24 (25, 50, 100, 200 and 400 nM) were complexed to Lipofectin for 24 hrsin Opti-MEM, and then for a further 67 hrs in complete media. Proteinsamples (30-40 μg of protein/lane) were analyzed by Western blotting asdescribed in the Material and Methods section, supra, with α-tubulinused as a control protein species. The % inhibition vs. control,untreated cells was determined by laser-scanning densitometry.

Example 24

[0321] Effect of PEG Oligonucleotides on PC3 Cell Growth

[0322] The effects of compounds 14 and compound 28 on the growth of PC3prostate cancer cells, in vitro, was also tested. PC3 cells were treatedwith oligonucleotide/Lipofectin complexes. As shown in FIG. 10,transfection of antisense oligonucleotide compound 14 at 400 and 200 nMstrongly inhibited cell growth, while compound 28 only slightly affectedthe proliferation rate.

Example 25

[0323] Effect of PEG Oligonucleotides on Production of Reactive OxygenSpecies in PC3 Cells

[0324] Production of reactive oxygen species or ROS in PC3 cells wasevaluated flow cytometrically by two methods. The first was based onoxidation of hydroethidium (HE) to ethidium (E), which subsequentlyintercalates into DNA with fluorescence detectable by flow cytometry.The second method employed the oxidation of cell-permeable2′,7′-dihydrodichlorofluorescein diacetate to fluorescent2′,7′-dichlorofluorescein (DCF). In PC3 cells, treatment with compound14/Lipofectin (400 nM/15 μg/ml) complexes for 24 hours in Opti-MEMgenerated ROS three days later as evaluated by flow cytometrically byboth E (1.9-fold increase vs. control, untreated cells) and DCF (2-foldincrease vs. control, untreated cells) fluorescence. As confirmed by thedata summarized by FIG. 11, compound 28 did not produced any increase inROS production vs. control, untreated cells. Furthermore, the productionof ROS is very closely linked to the rate of cellular proliferation;cells cease growing after treatment with 400 nM of compound 14, and thisoligonucleotide also causes an increase in the production of ROS (DCFand HE).

1 11 1 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 1 tctcccagcg tgcgccat 18 2 18 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 2tctcccagcg tgtgccat 18 3 18 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 3 atcctaagcg tgcgcctt 18 418 DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 4 tctcccagng tgngccat 18 5 17 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 5taccgcgtgc gaccctc 17 6 20 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 6 cagcgtgcgc catccttccc 207 35 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 7 cttttcctct gggaaggatg gcgcacgctg ggaga 35 820 DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 8 gatgcaccta cccagcctcc 20 9 33 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 9acggggtacg gaggctgggt aggtgcatct ggt 33 10 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 10acaaaggcat cctgcagttg 20 11 36 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 11 cccccaactg caggatgcctttgtggaact gtacgg 36

We claim:
 1. An oligonucleotide prodrug of the formula (I):

wherein: R₁ and R₂ are independently H or a polymer residue; L₁ and L₄are independently selected releasable linking moieties; L₂ and L₃ areindependently selected spacing groups; X₁ is a nucleotide or anoligonucleotide residue; m, n, o and p are independently zero or apositive integer, provided that either (o+n) or (p+m)≧2.
 2. The prodrugof claim 1, wherein said nucleotide is selected from the groupconsisting of

wherein M is O or S; B₁ and B₂ are independently selected from the groupconsisting of A (adenine), G (guanine), C (cytosine), T (thymine), U(uracil) and modified bases; R₁₀₀ and R₁₀₁, are independently selectedfrom the group consisting of H, OR′ where R′ is H, a C₁₋₆ alkyl,substituted alkyls, nitro, halo and aryl
 3. The prodrug of claim 1,wherein said oligonucleotide is contains from about 10 to about 1000nucleotides.
 4. The prodrug of claim 1, wherein M is S.
 5. The prodrugof claim 1, wherein the oligonucleotide is a phosphorthioateoligonucleotide.
 6. The prodrug of claim 1, wherein said oligonucleotideresidue is an antisense oligonucleotide residue or oligodeoxynucleotideresidue.
 7. The prodrug of claim 6, wherein said antisenseoligonucleotide residue or oligodeoxynucleotide residue is selected fromthe group consisting of, oligonucleotides and oligodeoxynucleotides withphosphorodiester backbones or phosphorothioate backbones, LNA, PNA,tricyclo-DNA, decoy ODN, RNAi, ribozymes, spiegelmers, and CpGoligomers.
 8. The prodrug of claim 6, wherein said antisenseoligonucleotide has a sequence selected from the group consisting of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, wherein X of SEQID NO: 4 is any compatible nucleotide.
 9. The prodrug of claim 1,wherein at least one of R₁ and R₂ is a polymeric residue having acapping group A, selected from the group consisting of OH, NH₂, SH,CO₂H, C₁₋₆ alkyls,

wherein X₂ and X₃ are independently selected nucleotide oroligonucleotide residues.
 10. A prodrug of claim 9, selected from thegroup consisting of:


11. The prodrug of claim 1 wherein L₄ is selected from the groupconsisting of:

wherein: Y₁₋₂₅ are independently selected from the group consisting ofO, S or NR₉; R₆₋₇, R₉₋₁₃, R₁₆₋₂₅, and R₂₇₋₄₁ are independently selectedfrom the group consisting of hydrogen, C₁₋₆alkyls, C₃₋₁₂ branchedalkyls, C₃₋₈ gcycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈ substitutedcyloalkyls, aryls, substituted aryls, aralkyls, C₁₋₆ heteroalkyls,substituted C₁₋₆heteroalkyls, C₁₋₆alkoxy, phenoxy and C₁₋₆ heteroalkoxy;Ar is a moiety which forms a multi-substituted aromatic hydrocarbon or amulti-substituted heterocyclic group; L₅₋₁₂ are independently selectedbifunctional spacers; Z is selected from among moieties activelytransported into a target cell, hydrophobic moieties, bifunctionallinking moieties and combinations thereof; c, h, k, l, r, s, v, w, v′,w′, c′, and h′ are independently selected positive integers; a, e, g, j,t, z, a′, z′, e′ and g′ are independently either zero or a positiveinteger; and b, d, f, i, u, q, b′, d′ and f′ are independently zero orone.
 12. The prodrug of claim 1 wherein L₁ is selected from the groupconsisting of:

wherein, Y_(1′-)Y_(25′) are independently selected from the groupconsisting of O, S or NR₉; R_(6′-7′), R_(9′-13′), R_(16′-25′), andR_(27′-41′) are independently selected from the group consisting ofhydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆substituted alkyls, C₃₋₈ substituted cyloalkyls, aryls, substitutedaryls, aralkyls, C₁₋₆ heteroalkyls, substituted C₁₋₆ heteroalkyls, C₁₋₆alkoxy, phenoxy and C₁₋₆heteroalkoxy; and L_(5′-12′) are independentlyselected bifunctional spacers.
 13. The prodrug of claim 1 wherein R₁₋₂are each polyalkylene oxides.
 14. The prodrug of claim 1 wherein R₁₋₂are each polyethylene glycols.
 15. The prodrug of claim 1 wherein R₁₋₂are independently selected from the group consisting of:J-O—(CH₂CH₂O)_(n′)— J-O—(CH₂CH₂O)_(n′)—CH₂C(O)—O—,J-O—(CH₂CH₂O)_(n′)—CH₂CH₂ NR₄₈—, J-O—(CH₂CH₂O)_(n′)—CH₂CH₂ SH,—O—C(O)CH₂—O—(CH₂CH₂O)_(n′)—CH₂C(O)—O—,—NR₄₈CH₂CH₂—O—(CH₂CH₂O)_(n′)—CH₂CH₂ NR₄₈—,—SHCH₂CH₂—O—(CH₂CH₂O)_(n′)—CH₂CH₂ SH—, wherein n′ is the degree ofpolymerization; R₄₈ is selected from the group consisting of hydrogen,C₁₋₆alkyls, C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆ substitutedalkyls, C₃₋₈ substituted cyloalkyls, aryls substituted aryls, aralkyls,C₁₋₆ heteroalkyls, substituted C₁₋₆heteroalkyls, C₁₋₆ alkoxy, phenoxyand C₁₋₆ heteroalkoxy; and J is a capping group.
 16. The prodrug ofclaim 1, wherein R₁₋₂ are independently —O—(CH₂CH₂O)_(x)— wherein x is apositive integer selected so that the weight average molecular weight isat least about 2,000 Da to about 136,000 Da.
 17. The prodrug of claim 1,wherein R₁₋₂ independently have a weight average molecular weight offrom about 3,000 Da to about 100,000 Da.
 18. The prodrug of claim 1,wherein R₁₋₂ independently have a weight average molecular weight offrom about 5,000 Da to about 40,000 Da.
 19. The prodrug of claim 8,wherein said antisense oligonucleotide is oblimersen (SEQ ID NO: 1). 20.An oligonucleotide prodrug of the formula:

wherein: L₂ is a spacing group; X₁ is a nucleotide or an oligonucleotideresidue; u′ is a positive integer; and T is a member of the groupconsisting of:

wherein: D′ is one of

and wherein R₆₁ is a polymer residue.
 21. A compound of claim 1 selectedfrom the group consisting of:

all of which comprise an oligonucleotide of SEQ ID NO:
 1. 22. A methodof making a prodrug comprising: reacting a compound of the formula:R₂-L₄-leaving group with a compound of the formula: H-L₃-X₁ underconditions sufficient to form a prodrug of the formula R₂-L₄-L₃-X₁,wherein: R₂ is a polymer residue; L₄ is a releasable linking moiety; L₃is a spacing group; X₁ is a nucleotide or an oligonucleotide residue.23. A method of treating a mammal, comprising administering to a mammalin need of such treatment an effective amount of a compound of claim 1.24. The method of claim 23, wherein the mammal is being treated forcancer.
 25. The method of claim 23, wherein X₁ is an antisenseoligonucleotide.
 26. The method of claim 23 wherein the mammal is alsotreated with a second anticancer agent that is administeredsimultaneously or sequentially with the oligonucleotide prodrug.